Adipose tissue lipoprotein lipase (LPL) hydrolyses the core of triglyceride-rich lipoproteins into free fatty acids. LPL is important for the generation of HDL2, and for the prevention of diabetic hypertriglyceridemia, and thus for the prevention of atherosclerosis. In previous studies, LPL could not be measured in human adipocytes, nor could the LPL protein be measured directly. The candidate has recenty developed a system of isolated human adipocytes that produce LPL in culture and is developing the techniques to measure LPL mass using specific antibodies. The specific aims of this project are as follows: 1) What is the pathway of LPL biogenesis? 2) Is LPL initially synthesized as an inactive or partially active precursor? 3) Is LPL regulated by such potential regulators as glucose, insulin, IGF1, adenosine, prostaglandin E2, and catecholamines? 4) What is the mechanism of such regulation? Because LPL cellular processing must first be characterized before mechanisms of regulation can be approached, aim number 4 represents a more long term goal. To approach question 1, LPL will be radiolabeled in the presence of each of the following inhibitors of glycoprotein processing: tunicamycin, CCCP, monensin, and colchicine. The LPL precursors that are formed will be identified by SDS-PAGE and autoradiography. To determine whether these precursors are bioactive (question 2), LPL activity will be measured. If a precursor is bioactive, this activity will be checked for the properties normally associated with LPL: salt inhibition, apo CII dependence, and an appropriate Km. In addition, specific activity will be determined by measuring LPL immunoreactive mass in an ELISA. To study regulation (question 3), LPL bio- and immunoactivity will be measured in cells exposed to each potential regulator listed above. To approach the mechanism of regulation (aim number 4), the processing inhibitors will be used to block the effect of each regulatory substance. In this way, the site of regulation can be isolated to a discrete location in glycoprotein processing.